The use of synthetic, short, single stranded oligonucleotide sequences to inhibit gene expression has evolved to the clinical stage in humans. It has been demonstrated that incorporation of chemically modified nucleoside monomers into oligonucleotides can produce antisense sequences which can form more stable duplexes and can have high selectivity towards RNA (Frier et al., Nucleic Acids Research, 1997, 25, 4429-4443). Two modifications that have routinely given high binding affinity together with high nuclease resistance are phosphorothioates and methylphosphonates.
There are a number of desirable properties such as specificity, affinity and nuclease resistance that oligonucleotides should possess in order to elicit good antisense activity. The ability to selectively target and be taken up by diseased cells is another important property that is desirable in therapeutic oligonucleotides. Natural oligonucleotides are polyanionic and are known to penetrate cells at very low concentrations. Neutral oligonucleotides, such as the methylphosphonates, are taken up by cells at much higher concentrations. Although the processes by which antisense oligonucleotides enter the cell membrane are not well understood, there is substantial evidence for distinct mechanisms of cell entry based on the electronic character of the antisense sequence.
Delivery of an antisense oligonucleotide to a specific, diseased cell is a very important area of active research. The majority of projected antisense therapies are for viral infections, inflammatory and genetic disorders, cardiovascular and autoimmune diseases and significantly, cancer. For example, in conventional chemotherapy, neoplasticity and virus-related infections are treated with high drug concentrations, leading to overall high systemic toxicity. This method of treatment does not distinguish between diseased cells and healthy ones.
In the treatment of cancers, the ability of antisense agents to down-regulate or inhibit the expression of oncogenes involved in tumor-transforming cells has been well documented in culture and animal models. For example, antisense inhibition of various expressed oncogenes has been demonstrated in mononuclear cells (Tortora et al., Proc. Natl. Acad. Sci., 1990, 87, 705), in T-cells, in endothelial cells (Miller et al., P.O.P., Biochimie, 1985, 67, 769), in monocytes (Birchenall-Roberts et al., Suppl. 1989, 13 (P.t. C), 18), in reticulocytes (Jaskulski, et al., Science 1988, 240, 1544)and in many other cell types, as generally set forth in Table 1.
TABLE 1 INHIBITION OF MAMMALIAN GENE EXPRESSION INHIBITION OF EXPRESSION CELL TYPE T cell receptor T cells Colony-stimulating factors Endothelial cells .beta.-Globin Reticulocytes Multiple drug resistance MCF-1 cells cAMP kinase HL-60 cells bcl-2 L697 cells c-myb Mononuclear cells c-myc T-lymphocytes Interleukins Monocytes
Virally infected cell cultures and studies in animal models have demonstrated the great promise of antisense and other oligonucleotide therapeutic agents. Exemplary targets from such therapy include eukaryotic cells infected by human immunodeficiency viruses(Matsukura et al., Proc. Natl. Acad. Sci., 1987, 84, 7706; Agrawal et al., Proc. Natl. Acad. Sci., 1988, 85, 7079), by herpes simplex viruses (Smith et al., P.O.P., Proc. Natl. Acad. Sci., 1986, 83, 2787), by influenza viruses (Zerial et al., Nucleic Acids Res., 1987, 15, 9909) and by the human cytomegalovirus (Azad et al., Antimicrob. Agents Chemother., 1993, 37, 1945). Many other therapeutic targets also are amenable to such therapeutic protocols.
The use of non-targeted drugs, to treat disease routinely causes undesirable interactions with non-diseased cells (Sidi et al., Br. J. Haematol., 1985, 61, 125; Scharenberg et al., J. Immunol., 1988, 28, 87; Vickers et al., Nucleic Acids Res., 1991, 19, 3359; Ecker et al., Nucleic Acids Res., 1993, 21, 1853). One example of this effect is seen with the administration of antisense oligonucleotide in hematopoietic cell cultures that exhibit non-specific toxicity due to degradative by-products.
Other research efforts suggest that antisense oligonucleotides possess more side effects in both in vitro and in vivo animal models. For example, non-complementary DNA sequences have been shown to interfere with cell proliferation and viral replication events through unknown mechanisms of action (Kitajima ibid) . This reinforces the desirability of oligonucleotides that are specifically targeted to diseased cells.
When phosphodiester oligonucleotides are administered to cell cultures, a concentration of typically about 1 mmol is required to see antisense effects. This is expected since local endonucleases and exonucleases cleave these strands effectively and only 1-2% of the total oligonucleotide concentration becomes cell-associated (Wickstorm et al., Proc. Natl. Acad. Sci. 1988, 85, 1028; Wu-Pong et al., Pharm. Res. 1992, 9, 1010). If chemically modified oligonucleotides, such as the phosphorothioates or methylphosphonates are used, the observed antisense effects are anywhere between 1 and 100 .mu.M. This observed activity is primarily due to the relatively slow cellular uptake of oligonucleotides. There is evidence which suggests that a 80 kiloDalton (kDa) membrane receptor mediates the endocytic uptake of natural and phosphorothioate oligonucleotides in certain type of cells. Other data question the existence of such a link between receptor-mediated oligonucleotide uptake and internalization of oligonucleotides. For example, inhibitors of receptor-mediated endocytosis have no effect on the amount of oligonucleotide internalized in Rauscher cells (Wu-Pong et al., Pharm. Res., 1992, 9, 1010). For uncharged methylphosphonates, it was previously believed that internalization of such agents occurred by passive diffusion (Miller et al., Biochemistry 1981, 20, 1874). These findings were disproved by studies showing that methylphosphonates take up to 4 days to cross phospholipid bilayers, which correlates well with the fate of internalization of natural oligonucleotides (Akhtar et al., Nucleic Acids Res., 1991, 19, 5551).
Increased cellular uptake of antisense oligonucleotides by adsorptive endocytosis can be obtained by liposome encapsulation. In one study, researchers showed that a 21-mer complementary to the 3'-tat splice acceptor of the HIV-1 was able to markedly decrease the expression of a p24 protein while encapsulated into a liposome containing diastearoylphosphatidylethanol-amine (Sullivan et al., Antisense Res. Devel., 1992, 2, 187). Many other examples have been reported, including pH-sensitive liposomes (Huang et al., Methods Enzymol., 1987, 149, 88) which are well detailed in several good review articles (Felgner et al., Adv. Drug Delev. Rev., 1990, 5, 163 and Farhood et al., N.Y. Acad. Sci., 1994, 716, 23). When phosphorothioate oligonucleotides, that are complementary to the methionine initiation codon of human intracellular adhesion molecule-1, were encapsulated, a 1000-fold increase of antisense potency was seen relative to the non-encapsulated phosphorothioate oligonucleotide (Bennett et al., Mol. Pharmacol., 1992, 41, 1023). The oligonucleotide delivery systems are good for in vitro cell systems, but have not been shown to be widely applicable to in vivo studies, due to rapid liposome destabilization and non-specific uptake by liver and spleen cells.
Other, non-specific oligonucleotide uptake enhancements attend attaching hydrophobic cholesterol (Letsinger et al., Proc. Natl. Acad. Sci., 1989, 86, 6553) type or phospholipid type molecules (Shea et al., Nucleic Acids Res., 1990, 18, 3777) to the oligonucleotides. It has been shown that the coupling of a single cholesterol moiety to an antisense oligonucleotide increases cellular uptake by 15-fold (Boutorin et al., FEBS Lett., 1989, 254, 129). When the cationic polymeric drug carrier poly(L-lysine) is conjugated to oligonucleotide sequences, a marked increase of non-specific oligonucleotide cellular uptake occurs (Lemaitre et al., Proc. Natl. Acad. Sci., 1987, 84, 648; Leonetti et al., Gene 1988, 72, 323; Stevenson et al., J. Gen. Virol., 1989, 70, 2673). This cationic polymer has been used to deliver several types of drugs with cellular uptake mediated by an endocytic-type mechanism. However, the high molecular weight polylysine is cytotoxic even at low concentrations.
Cell surface receptors are good candidates to serve as selective drug targets. The presence of specific receptors implies that natural endogenous ligands are also present. It is the complexation of the ligand with the appropriate receptor that elicits a cascade of cellular events leading to a desired function. An oligonucleotide drug linked to such an endogenous ligand or a synthetic ligand of equal affinity towards the receptor in question, is considered "targeted" to the receptor.
The potential of carbohydrate drug targeting has become increasingly apparent (Shen et al., N.Y. Acad. Sci., 1987, 507, 272; Monsigny et al., N.Y. Acad. Sci., 1988, 551, 399; Karlsson et al., TIPS 1991, 12, 265) as an alternate method for site-specific drug delivery. Complex carbohydrates are involved in many cellular recognition processes such as adhesion between cells, adhesion of cells to the extracellular matrix, and specific recognition of cells (Ovarian egg with sperm) by one another (Yamada, K. M., Annu., Rev. Biochem., 1983, 52, 761; Edelman, G. M., Annu. Rev. Biochem., 1985, 54, 135; Hook et al., Annu. Rev. Biochem., 1984, 53, 847; Florman, H. M., Cell, 1985, 41, 313. It is also known that the concentrations of various glycosylated proteins that circulate in the blood are constantly regulated by cells in various tissues. Nature controls and regulates such diverse functions with the aid of specific proteins appearing at the surface of various cells, which have the ability to decode the information found in complex carbohydrate structures. These proteins are collectively called lectins and act as receptors for carbohydrates (Goldstein et al., Nature, 1980, 285, 66). Many endogenous lectins are expressed at the surface of normal and malignant cells and are involved in many poorly understood biological processes.
The structural information obtained from a large number of mammalian lectins has led to their classification into several families.
TABLE 2 PROPERTIES OF C-TYPE AND S-TYPE ANIMAL LECTINS PROPERTY C-TYPE LECTINS S-TYPE LECTINS Ca.sup.++- dependance Yes No Solubility Variable Buffer soluble Location Extracellular Intracellular/extra State of cysteines Disulfides cellular Carbohydrate Different types Free thiols specificity Mostly .beta.- galactosides
The C-lectins or calcium-dependent lectins possess carbohydrate recognition domains (CRDs) of the 115-134 amino acids which contain 18 highly conserved and 14 invariant residues (Drickamer, K., J. Biol. Chem., 1988, 263, 9557; Drickamer, K., Curr. Opin. Struc. Biol., 1993, 3, 393; Drickamer, K., Biochemical Society Transactions, 1993, 21, 456). The C-lectins are interesting from a pharmacological point of view since they recognize specific carbohydrates and immediately endocytose the receptor-bound glycoprotein complex via coated pits and vesicles.
These vesicles which are also referred to as endosomes, bring the receptor-glycoprotein complex to other cellular compartments, called the lysozomes, where protein degradation occurs (Breitfeld et al., Int. Rev. Cytol., 1985, 97, 4795). The range of C-type lectin carbohydrate specificity differs form cell to cell and from tissue to tissue.
The first membrane lectin was characterized on hepatocyte liver cells (Van Den Hamer et al., J. Mol. Biol., 1970, 245, 4397). The hepatic asialoglycoprotein receptor (ASGP-R) was isolated by Ashwell and Harford (Ashwell, G.; Herford, J., Ann. Rev. Biochem. 1982, 51, 531; Schwartz, A. L., CRC Crit. Rev. Biochem ., 1984, 16, 207). These lectins internalized efficiently and cleared plasma levels from ceruloplasmin which contained abnormally truncated N-oligosaccharides lacking the terminal sialic acid residues. Other artificial molecules which have terminal galactose or N-acetylgalactosamine residues have been found to bind with high affinity to this lectin. This unique specificity between the exposed galactose units and the ASGP-R suggested the design and testing of glycotargeting systems and the use of lectins as specific drug delivery targets.
TABLE 3 MEMBRANE SPANNING C-TYPE LECTINS NAME TISSUE SUGAR SPECIFICITY ASGP-R (type II) Liver Hepatocytes Galactose and N-acetylgalactoseamine.sup.1 Placental Placenta Fucose and mannose.sup.2 (type receptor II) Macrophage Liver Kupffer Galactose and receptor cells type II) N-acetyl galactoseamine Kupffer cell Liver Kupffer Galactose and fucose receptor cells (type II) IgE Fc receptor B cells Galactose.sup.5 (type II) P-selectin Platelets Fucose and sialic acid (type IV) E-selectin Endothelial cells Fucose and sialic (type (type IV) IV) acid.sup.7 L-selectin Leukacytes Fucose and sialic acid.sup.8 (type IV) Mannose receptor Macrophages Mannose and fucose.sup.9 (type VI) .sup.1 Spiess et al., J. Biol. Chem., 1985, 260, 1979. .sup.2 Curtis et al., Proc. Natl. Acad. Sci., 1992, 89, 8356. .sup.3 Ii, et al., J. Biol. Chem., 1990, 265, 11295. .sup.4 Hoyle et al., J. Biol. Chem., 1988, 263, 7487. .sup.5 Kikutani et al., Cell, 1986, 47, 657. .sup.6 Johnston et al., Cell, 1989, 56, 1033. .sup.7 Bevilacqua et al., Science, 1989, 243, 1160. .sup.8 Laskyk et al., Cell, 1989, 56, 1045. .sup.9 Taylor et al., J. Biol. Chem., 1992, 267, 1719.
Many other cell lines, some summarized in Table 2, have surface carbohydrate-type receptors that mediate uptake of various ligands (Drickamer, K., Cell 1991, 67, 1029). Immune cells like monocytes and macrophages possess a number of surface glycoproteins that enable them to interact with invading micro-organisms (Gordon et al., J. Cell Sci. Suppl., 1988, 9, 1). Drugs need to be carried to target cells via a carrier or high affinity ligand which is attached to the drug. The different carriers for glycotargeting can be glycoproteins or neoglycoproteins, (glycopeptides or neoglycopeptides) and as glycosylated polymers.
The in vitro glycotargeting principle is relatively simple, but its in vivo applicability is difficult. Synthetic efforts have generated liposomes (also referred as immunoliposomes) and polylysine carriers, in which antibodies and some carbohydrate conjugate ligands have been covalently attached on the outer bilayer. For example, when natural oligonucleotides complementary to the translation initiation region of VSV N protein mRNA were encapsulated with liposomes whose outer membrane had several H2K-specific antibodies to L929 cells, there was a marked decrease in viral replication only within L929 infected cells (Leonetti et al., Proc. Natl. Acad. Sci., 1990, 87, 2448). Receptor-mediated endocytic mechanisms have been exploited by attachment of cell-specific ligands and antibodies to polylysine polymers. For example, c-myb antisense oligonucleotides conjugated with polylysine-folic acid (Citro et al., Br. J. Cancer 1992, 69, 463) or polylysine-transferrin (Citro et al., Proc. Natl. Acad. Sci., 1992, 89, 7031) targets were found to better inhibit HL-60 leukemia cell line proliferation than oligonucleotides without conjugated carriers. Another promising polylysine-asialoorosomucoid carrier was conjugated with phosphorothioate oligonucleotides complementary to the polyadenylation signal of Hepatitis B virus (Wu, G. Y.; Wu, C. H., J. Biol. Chem., 1992, 267, 12436).
Methods have been previously developed that utilize conjugates to enhance transmembrane transport of exogenous molecules. Ligands that have been used include biotin, biotin analogs, other biotin receptor-binding ligands, folic acid, folic acid analogs, and other folate receptor-binding ligands. These materials and methods are disclosed in U.S. Pat. No. 5,108,921, issued Apr. 28, 1992, entitled "Method for Enhanced Transmembrane Transport of Exogenous Molecules", and U.S. Pat. No. 5,416,016, issued May 16, 1995, entitled "Method for Enhancing Transmembrane Transport of Exogenous Molecules", the disclosures of which are herein incorporated by reference.
These immunoliposomes and antibody-polymer targeting exhibited no in vivo activity. With similar drawbacks as their non-specific counterparts, the immunoliposome-drug complexes are mostly immunogenic and are phagocytosed and eventually destroyed in the lysosome compartments of liver and spleen cells. As for the antibody-polylysine-drug complexes, they have shown substantial in vitro cytotoxic activity (Morgan et al., J. Cell. Sci., 1988, 91, 231). Other carriers are glycoproteins. On such large structures, a few drug molecules can be attached. The glycoprotein-drug complexes can subsequently be desilylated, either chemically or enzymatically, to expose terminal galactose residues.
Glycoproteins and neoglycoproteins are recognized by lectins such as the ASGP-R. Glycoproteins having a high degree of glycosylation heterogeneity are recognized by many other lectins making target specificity difficult (Spellman, N. W., Anal. Chem., 1990, 62, 1714). Neoglycoproteins having a high degree of homogeneity exhibit a higher degree of specificity for lectins especially the ASGP-R. Many experimental procedures which are used to couple sugars to proteins have been reviewed by Michael Brinkley (Brinkley, M., Bioconjugate Chem., 1992, 3, 2). These neoglycoproteins may mimic the geometric organization of the carbohydrate groups as in the native glycoprotein and should have predictable lectin affinities. Successful in vitro delivery of AZT-monophosphate, covalently attached to a human serum albumin containing several mannose residues was achieved in human T4 lymphocytes (Molema et al., Biochem. Pharmacol., 1990, 40, 2603).
Examples of antisense oligonucleotide-neoglycoprotein complexes have been previously reported (Bonfils et al., Nucleic Acids Res., 1992, 20, 4621). The authors mannosylated bovine serum albumin and attached, covalently from the 3'-end, a natural oligonucleotide sequence. The oligonucleotide-neoglycoprotein conjugate was internalized by mouse macrophages in 20-fold excess over the free oligonucleotide. Biotinylated oligonucleotides, were also disclosed which were non-covalently associated with mannosylated streptavidin (Bonfils et al., Bioconjugate Chem., 1992, 3, 277). Such complexes were also better internalized by macrophages. Other successful examples consisted of antisense oligonucleotides which were noncovalently associated with asialoglycoprotein-polylysine conjugates. Such oligonucleotide conjugates were found to internalize more efficiently into hepatocytes (Bunnel et al., Somatic Cell Molecular Genetics, 1992, 18, 559; Reinis et al., J. Virol. Meth., 1993, 42, 99) and into hepatitis B infected HepG2 cells (Wu, G. Y.; Wu, C. H., J. Biol. Chem., 1992, 267, 12436).
Polymeric materials have been assessed as drug carriers and three of them, dextrans, polyethyleneglycol (PEG) and N-(2-hydroxypropyl)methacrylamide (HMPA) co-polymers, have been successfully applied in vivo (Duncan, R., Anticancer Drugs, 1992, 3, 175). This research has been focused mainly at treatments for cancer and as a requisite the size of the compounds are between 30-50 kDa to avoid renal excretion (Seymour, L. W., Crit. Rev. Ther. Drug Carrier Syst., 1992, 9, 135).
In order to examine the chemistry and related methodologies involving the preparation of glycoprotein-drug and neoglycoprotein-drug glycoconjugates, certain groups have investigated the use of simpler high affinity ligands for specific drug delivery. Initially, several sugars were attached to small peptides in an attempt to obtain mimics of multivalent N-linked oligosaccharides (Lee, R. T., Lee, Y. C., Glycoconjugate J., 1987, 4, 317; Plank et al., Biconjugate Chem., 1992, 3, 533; Haensler et al., Bioconjugate Chem., 1993, 4, 85).
Other groups investigated the use of sugar clusters lacking a protein backbone and eventually used low molecular weight N-linked oligosaccharides with a minimum carbohydrate population to bind with high affinity to lectins as the ASGP-R. Branched N-linked oligosaccharide-drug conjugates can be used instead of neoglycoprotein-drug complexes. The total synthesis of branched N-linked oligosaccharides is still a difficult task, however they could be obtained by enzymatic cleavage from protein backbones (Tamura et al., Anal. Biochem., 1994, 216, 335). This method requires expensive purifications and only generates low quantities of chemically defined complex oligosaccharides. The affinity of N-linked oligosaccharide clusters towards many lectins has been demonstrated and has helped researchers to locate different new mammalian lectins in animals (Chiu et al., J. Biol. Chem., 1994, 269, 16195).
One process of increasing the intracellular oligonucleotide concentration is via receptor-mediated endocytic mechanisms. This novel drug targeting concept has been demonstrated in vitro by several groups. Oligonucleotides have been attached to glycoproteins, neoglycoproteins and neoglycopolymers possessing a defined carbohydrate population which, in turn, are specifically recognized and internalized by membrane lectins. To the best of our knowledge in vivo applicability of oligonucleotide-carbohydrate conjugates has not been previously demonstrated.
It has also been shown in in vitro experiments that synthetic neoglycoproteins containing galactopyranosyl residues at non-reducing terminal positions are recognized by the ASGP-R with increasing affinity as the number of sugar residues per molecule is increased (Kawaguchi et al., J. Biol. Chem. 1981, 256, 2230).